The long-term objective of the present research is to elucidate the roles of certain factors that regulate mineralized tissue formation and to contribute to the development of therapies for mineralized tissue disorders. S100A4 is a calcium-binding protein that has been implicated in cancer metastasis, however, recent evidence has shown that S100A4 is also expressed by normal cells such as osteoblastic cells. However, prior to the initiation of mineralized matrix formation, the level of S100A4 expression is markedly diminished. Inhibition of S100A4 protein synthesis by an antisense approach in MC3T3E1-derived osteoblastic cell clones significantly accelerated and increased the formation of mineralized nodules in vitro. Furthermore those clones expressed bone sialoprotein protein osteocalcin, markers associated with matrix mineralization and osteoblastic phenotype, at the stage when they were absent from the control cells. Here we hypothesize that S100A4 functions as a negative regulator of mineralization likely by modulating osteoblast differentiation. To address this hypothesis, the following specific aims are proposed: 1. To determine the expression levels of Cbfa1 and osteoblastic phenotypic markers for early, intermediate, and late stages of cell differentiation of MC3T3E1-derived cell clones expressing low levels of S100A4 (inhibited by S100A4 antisense). 2. To over-express S100A4 in MC3TE1 pre-osteoblastic cells. 3. To evaluate the ability of MC3T3E1-derived cell clones over- expressing S100A4 to form mineralized nodules in vitro and determine the expression of Cbfa and osteoblastic phenotypic markers for early, intermediate, and late stages of cell differentiation. 4. To transplant the cell clones expressing low levels of S100A4 and those over-expressing S100A4 into immunodeficient mice and evaluate their capabilities to form mineralized matrices in vivo. The combination of this in vitro/in vivo approach would provide insights into the potential regulatory roles of S1004A4 in osteoblast differentiation and biomineralization.